Two Distinct Hemolytic Activities in Xenorhabdus nematophila Are Active against Immunocompetent Insect Cells

The genus Xenorhabdus consists of the specific bacterial symbionts of the entomopathogenic nematodes of the family Steinernematidae (40) and was separated from the genus Photorhabdus (11), which contains the symbionts of the entomopathogenic nematodes of the family Heterorhabditidae. Both genera are entomopathogenic gram-negative bacteria belonging to the Enterobacteriaceae. The nematodes carry their bacterial symbionts monoxenically in a special vesicle of the infective stage (L3 juveniles) in Steinernematidae (8) and throughout the whole intestine of Heterorhabditidae (20). These bacteria are transported by their nematode hosts into the hemocoel of the insect prey, which is killed, probably via a combination of toxin action and septicemia. The bacterial symbionts also contribute to the symbiotic relationship by establishing and maintaining suitable conditions for nematode reproduction (31). Recently, isolation of some Photorhabdus strains from infected humans in Australia and the United States was reported (21, 30), and the strains from the United States were classified as Photorhabdus asymbiotica (23). The form of the bacterium that is normally isolated from symbiotic infective-stage nematodes is referred to as phase I. Like many pathogenic bacteria, Xenorhabdus and Photorhabdus strains spontaneously produce colonial variants which have been called phase II variants (10). The two variants of the bacteria have generally been shown to be equally pathogenic for the larvae of the greater wax moth, Galleria mellonella (3). However, Volgyi et al. (42) described for the first time a phase II variant that showed reduced virulence in the tobacco hornworm, Manduca sexta. Xenorhabdus nematophila and Photorhabdus luminescens are highly pathogenic to insects, and 50% insect mortality has been reported with direct infection with fewer than 20 bacteria per larva (5). The bacterial factors involved in killing of the insect or in overcoming the insect immune reactions are still under investigation. Following invasion of the insect host by the nematodes, both bacteria produce potential virulence factors, including lipase, protease, lecithinase, and lipopolysaccharides (LPSs), in the hemocoel (for a review, see reference 24). It was shown that purified LPS, Photorhabdus protease fractions, or Xenorhabdus lecithinase isomers showed no toxic effect following injection into insect hemocoel (12, 16, 39). Recently, a novel toxin complex with both oral and injectable activities against a wide range of insects was identified in a supernatant of P. luminescens (13). Purified toxin complex a (Tca) has specific effects on the midgut epithelium of the insect (9). In order to study Xenorhabdus and Photorhabdus virulence in insects, a genetic approach was also used. Avirulent mutants of X. nematophila have been isolated by transposon mutagenesis (Tn5). These mutants were pleiotropic, but all five mutants that tested as avirulent in G. mellonella were nonmotile and partially impaired in blood hemolysis (43). It was also shown that a homoserine lactone autoinducer restored virulence to one avirulent X. nematophila strain and stimulated the level of bacterial lipase activity (17). Recently we reported that flhDC, the flagellar master operon of X. nematophila, controls flagellin expression. Furthermore we revealed that lipolytic and extracellular hemolysin activity is flhD dependent. We also showed that the flhD null mutant displayed an attenuated virulence phenotype in the common cutworm, Spodoptera littoralis, compared to the wild-type strain (25). The recently published partial genome sequence of P. luminescens (22) revealed a diverse array of genes that putatively encodes potential virulence factors. These factors include exoenzymes (proteases, lipases, and chitinases), a type III secretion system (Yop homolog), and several classes of toxins (insecticidal toxin complex, Rtx-like toxins, and hemolysin and cytotoxin homologs) (22). Until now, studies examining hemolytic activity of both genera have not been reported. Cytolysins are proteins which cause lysis of red blood cells (RBC) as well as nucleated cell types by hydrolysis (lipases, phospholipases, or proteases) or by forming pores in the plasma membrane. Surfactants may also cause cytolysis by solubilization of the target cell membrane. Bacterial cytolysins are usually recognized as hemolysin on blood agar where a transparent zone appears around colonies. The production by a few strains of Xenorhabdus and Photorhabdus of hemolysin has been detected on agar supplemented with sheep blood (6, 21, 25). Apart from their phoretic location inside infective juvenile nematodes, these bacteria are only observed in insect hemolymph, where they enter their growth cycle. Here the bacteria are in contact with hemocytes which achieve defense reactions in insects. Some of these cells are immunocompetent cells able to engulf (phagocytosis) or to isolate and kill (nodule formation) bacteria. We hypothesize that hemolytic activities could target the immunocompetent cells in insect hemolymph. In this study, we report that different cytolytic activities were found in supernatants of Xenorhabdus whereas none was detected in supernatants of various strains of Photorhabdus. We have studied the kinetics of the production of cytolytic activities over the course of in vitro bacterial growth. We also provide evidence on the characteristics and on the specificity of each of these cytolytic activities against mammalian RBC and insect hemocyte types.